Development of a dot blot assay for the rapid detection of central nervous system tissue on meat and contact surfaces

As a potential transmitter of bovine spongiform encephalopathy (BSE), tissue from bovine central nervous system (CNS) is not accepted in meat and meat products. Western blot analysis of the CNS marker myelin proteolipid protein (PLP) detects CNS contamination selectively and sensitively. In this study, a rapid dot blot assay using an anti-PLP antibody was developed to screen CNS contamination of meat and contact surfaces. The detection limit was 0.01% bovine brain in minced bovine muscle. When applied to a swab test, down to 0.5 mg of CNS tissue on meat or other surfaces was detectable. Other offal tissues or peripheral nerves did not interfere with the assay. The test allows a differentiation between mammalian and avian CNS but not among mammalian species. The swab test was applied immediately after slaughtering at several areas of the bovine head. CNS was not detectable at any region which may enter the food chain.     

牛肉中疯牛病特殊风险物质荧光RT-PCR检测方法的建立

本研究建立了检测牛肉中疯牛病特殊风险物质（主要是中枢神经组织）标记物GFAP mRNA的荧光RT-PCR检测技术。试验结果表明：该技术具有良好的种属特异性和组织特异性，只能从牛源和羊源的中枢神经组织中检测到GFAP，猪源和禽源检测结果为阴性，而且只有脑和脊髓等中枢神经组织产生阳性反应，其他内脏组织以及不同部位牛肉检测结果均为阴性；敏感性检测结果表明，该方法最低检测限达到0.001%以下；稳定性试验结果表明，100°C 加热处理30min对检测结果无明显影响，中枢神经组织在30°C以上室温可以稳定存放4天， 在4°C可以稳定冷藏2周，检测结果仍然为阳性。该结果显示，所建立的荧光RT-PCR技术用于牛肉中疯牛病特殊风险物质的检测具有特异性强、敏感性高、稳定性好及快速方便等优点，适合于在日常检测工作中推广使用。     

Myelin proteolipid protein (PLP) as a marker antigen of central nervous system contaminations for routine food control

Spreading transmissible spongiform encephalopathies (TSE) have been widely attributed to transmission by ingestion of mammalian central nervous system (CNS) tissue. Reliable exclusion of this epidemiological important route of transmission relies on an effective surveillance of food contamination. Here, myelin proteolipid protein (PLP) is identified as a specific and largely heat-resistant marker for detection of food contaminations by CNS tissue. PLP is a component of oligodendritic glial sheaths of neuronal processes that is specifically expressed in the CNS. A highly selective polyclonal antibody was developed directed against an epitope present in the full-length PLP protein, but absent from the developmentally regulated splice variant DM-20. In combination with a hydrophobic extraction of PLP from tissue samples, the antibody reliably detected PLP from spinal cord, cerebellum, and cortex of different mammalian species. Consistent with earlier reports on PLP expression, no cross-reactivity was observed with peripheral nerve or extraneural tissue, except for a very faint signal obtained with heart. When applied to an artificial CNS contamination present in sausages, the antibody reliably detected a low concentration (1%) of the contaminant. Application of heat, as used during conventional sausage manufacturing, led to a predominant alteration of arginine residues in the PLP protein and a partial loss of immunoreactivity. In contrast, a stretch of hydrophilic amino acids(112-122) proved to be heat-resistant, preserving the immunogenicity of this PLP epitope during heating. Taken together, the excellent CNS specificity of PLP immunodetection and the presence of a heat-resistant epitope have permitted the development of a highly sensitive immunoassay for CNS contamination in routine food control.     

表面增强拉曼光谱快速检测生鲜肉中的瘦肉精

为了快速检测生鲜肉中的瘦肉精,该研究利用表面增强拉曼光谱技术,以沙丁胺醇为检测目标物,建立了一种快速检测肌肉组织和肝脏中瘦肉精含量的方法。在碱性环境下利用乙酸乙酯对样品中沙丁胺醇进行提取,采用Savitzky-Golay 5点平滑法和自适应迭代重加权惩罚最小二乘法消除光谱噪声以及荧光背景对分析建模的影响。为检测方法的重复性,对50个相同沙丁胺醇质量分数(1 mg/kg)的肌肉组织样品进行信号采集,对沙丁胺醇特征峰强度进行分析,621、814、1 253、1 489、1 609 cm~(-1) 5个特征峰强度的相对标准偏差(RSD)为6.54%、6.07%、8.65%、7.44%、6.81%,说明该方法具有较好的重复性。建立沙丁胺醇标准溶液的预测模型,沙丁胺醇浓度与其特征峰强度相关性较好,决定系数R~2为0.968。对肌肉组织和肝脏中沙丁胺醇含量进行检测,检测范围分别为0.01~5和0.02~5 mg/kg,检出限分别为0.01和0.02 mg/kg,其含量与预测实测值决定系数为0.912和0.921。研究表明,该方法可以实现肌肉组织和肝脏中沙丁胺醇含量的定量预测。     

Real-time PCR技术用于中国大菱鲆虹彩病毒的组织敏感性检测及病毒流行情况调查

中国大菱鲆虹彩病毒(turbot reddish body iridovirus, TRBIV)是一种感染养殖大菱鲆的鱼类虹彩病毒,它可以引起大菱鲆病毒性红体病并导致养殖大菱鲆大量死亡。本研究利用TRBIV主要衣壳蛋白基因序列设计的一对引物,结合内嵌式核酸染料SYBR Green І,建立了TRBIV特异的Real-time PCR检测方法。实验结果表明,该对引物具有较高的灵敏度和较强的特异性,能够检测相当于102数量级的TRBIV基因组拷贝,而不与健康大菱鲆组织DNA、淋巴囊肿病毒DNA发生交叉反应。最后,应用建立的Real-time PCR检测方法,开展了TRBIV的组织敏感性检测和病毒流行情况调查。结果发现,大菱鲆的脾脏、肾脏、脑、鳃、心脏、肝脏、消化道、血液等组织中均可检测到TRBIV的存在,其中脾和肾是TRBIV的最主要的靶器官,每毫克组织的病毒含量分别高达5.23106个和2.18106个。分子流行病学调查结果显示,在山东半岛的多个大菱鲆养殖场中均存在TRBIV的感染和流行。     

Chemical imaging of microstructures of plant tissues within cellular dimension using synchrotron infrared microspectroscopy

Synchrotron radiation-based Fourier transform infrared microspectroscopy (SR-FTIR) is an advanced bioanalytical technique capable of exploring the chemistry within microstructures of plant and animal tissues with a high signal to noise ratio at high ultraspatial resolutions (3-10 microm) without destruction of the intrinsic structures of a tissue. This technique is able to provide information relating to the quantity, composition, structure, and distribution of chemical constituents and functional groups in a tissue. The objective of this study was to illustrate how the SR-FTIR technique can be used to image inherent structures of plant tissues on a cellular level (pixel size, approximately 10 microm x 10 microm). The results showed that with the extremely bright synchrotron light, spectra with high signal to noise ratios were obtained from areas as small as 10 microm x 10 microm in the plant tissue, which allowed us to "see" plant tissue in a chemical sense on a cellular level. The ultraspatial resolved imaging of plant tissues by stepping in pixel-sized increments was obtained. Chemical distributions of plant tissues such as lignin, cellulose, protein, lipid, and total carbohydrate could be mapped. These images revealed the chemical information of plant intrinsic structure. In conclusion, SR-FTIR can provide chemical and functional characteristics of plant tissue at high ultraspatial resolutions. The SR-FTIR microspectroscopic images can generate spatially localized functional group and chemical information within cellular dimensions.     

用于污染黄曲霉毒素花生分选的荧光信号研究

为在加工前将黄曲霉毒素超限的带衣花生米从原料中剔除,参照已有的色选系统,提出一种依据黄曲霉毒素含量超限带衣花生米的专属荧光信号进行逐粒分选的技术构想。采用Cary Eclipse荧光分光光度计测定100粒外观具有代表性的带衣花生米表面的紫外-荧光规律,通过与免疫亲和层析净化荧光光度法(GB/T18979-2003)检测结果对比,判定了黄曲霉毒素超限带衣花生米的荧光光谱特征;通过绘制450/490、460/490荧光强度比值的箱线图,评估了表面荧光法判断黄曲霉毒素超限带衣花生米的准确率;在搭建的荧光成像系统上,对黄曲霉毒素超限带衣花生米进行了荧光成像。检测发现,在365 nm波长激发下,黄曲霉毒素超限带衣花生米在420~460 nm处有荧光峰;以450/490荧光强度比值为依据剔除超限值带衣花生米的判断准确率为81%;a.u.40的带衣花生米可在图像中呈现亮蓝荧光光斑。表明表面荧光信号可作为带衣花生米在线、无损、逐粒分选的专属光学信号,用于黄曲霉毒素超限带衣花生米的剔除。     

Application of a real time polymerase chain reaction method to detect castor toxin contamination in fluid milk and eggs

The castor seed contains ricin, which is one of the most potent biological toxins and is widely considered to be a threat agent for bioterrorism. In this study, a rapid and sensitive PCR method was applied to the detection of castor contamination in milk and liquid egg samples. The targeting gene sequence of the primer set, Ricin-F4/R4, was not found in either the bovine or chicken genome. Primers against a highly conserved sequence from the 18S ribosomal RNA gene were used as a positive control for DNA extraction and PCR reaction efficiency. The quantity and quality of DNA prepared from castor spiked or nonspiked milk and egg samples obtained from three different DNA extraction methods were compared. The cetyl trimethylammonium bromide (CTAB) method yielded the highest quality of DNA and is most suitable for the sensitive detection of castor DNA by real-time PCR in both milk and liquid egg matrixes. However, taking time and cost into consideration, a commercial kit designed for extraction of DNA from stool samples could be used as an alternative method for the routine extraction of DNA from milk for real-time PCR assays. The egg matrix was found to inhibit PCR amplification and interfere with two of the three methods tested for DNA extraction. Egg yolk had a greater negative effect on PCR amplification than the egg white matrix. Our results affirm the necessity of performing individual validations for each food matrix. Both real-time PCR systems used in this study, TaqMan and SYBR Green I dye, were capable of detecting 100 ng of castor acetone powder, corresponding to 5 ng of ricin, in 1 mL of milk or liquid egg, well below the toxic dose for humans. On the basis of these results, the real-time PCR method for detection of intentional castor contamination is applicable to milk and egg matrixes.     

Sandwich enzyme-linked immunosorbent assay for the detection of bovine blood in animal feed

The feeding of ruminant proteins to ruminants is prohibited in most countries because the practice is thought to be responsible for the spread of bovine spongiform encephalopathy. However, currently available methods to detect ruminant blood products in rendered feedstuffs are inadequate because they lack species specificity, tissue specificity, and are not based on a thermostable analyte. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for this study that provides reliable and sensitive (0.05-0.5% v/v) detection of bovine blood materials in animal feed. The new sandwich ELISA employs two previously developed monoclonal antibodies (MAbs), Bb6G12 as the capture antibody and biotinylated MAb Bb3D6 as the detecting antibody, and is bovine-specific and blood-specific. The assay is based on the detection of a 60 kDa thermostable protein in bovine blood and provides a useful regulatory tool for monitoring fraudulent labeling or contamination of bovine blood in both heat-processed feedstuffs and unprocessed raw materials. Keywords: Bovine; blood; monoclonal antibody; sandwich ELISA.     

Steady-state and time-resolved spectroscopy of F420 extracted from methanogen cells and its utility as a marker for fecal contamination

Methanogenic bacteria, which are common inhabitants of the animal digestive tract, contain the fluorescent compound F420 (coenzyme 420), a 7,8-didemethyl-8-hydroxy-5-deazariboflavin chromophore. F420 was characterized as an initial step in determining if this compound would be useful as a fluorescent marker for the detection of fecal and ingesta contamination. Using a single anion exchange chromatographic process, F420 was separated from other cell components of a Methanobrevibacter sp. cell culture. The extent of separation was determined spectroscopically. To aid in the development of possible techniques for the detection of fecal contamination using F420 as a marker, further spectroscopic investigation of F420 was conducted using steady-state and time-resolved fluorescence methods. The fluorescence lifetime of F420 in an elution buffer of pH 7.5 was found to be 4.2 ns. At higher pH values, the fluorescence decay, F(t), was best described by a sum of two exponentials: at pH 13, F(t) = 0.31 exp(-t/4.20 ns) + 0.69 exp(-t/1.79 ns). Further investigation using front-faced fluorescence techniques has shown that emission from F420 can be collected efficiently from samples of methanogen cell cultures as well as from fecal material.     

Detection of castor contamination by real-time polymerase chain reaction

Due to the potential for intentional contamination of food with crude preparations containing ricin, a real-time PCR method was developed for the detection of castor plant material in ground beef. One primer pair was identified and confirmed to be castor-specific and efficient for amplification of ricin in DNA extracts from castor or beef matrices. Of three different DNA extraction protocols compared, the hexadecyltrimethylammonium bromide (CTAB) method yielded the highest quality of DNA for QPCR assay. The detection limit for castor contamination in ground beef samples was <0.001% (<10 microg of castor acetone powder per gram of beef, corresponding to 0.5 microg of ricin), indicating excellent sensitivity for the assay, well below the threshold for oral toxicity.     

牛皮肤成纤维细胞的分离培养与人溶菌酶基因的转染研究

摘要：为得到转染人溶菌酶基因的核供体细胞,采用组织块贴壁法分离培养牛耳成纤维细胞,经传代纯化培养,绘制生长曲线,将纯化好的带有人溶菌酶和绿色荧光标记基因的载体用脂质体法转入第4代成纤维细胞。24 h后荧光显微镜下检测到有绿色荧光蛋白表达,经500mg/ml G418筛选2周后,G418减半继续培养2～3代 ,PCR检测到有目的条带,说明目的基因已成功导入,因此本研究中获得的转基因牛耳成纤维细胞可能作为体细胞核移植的供体细胞进行转基因克隆牛研究。     

Quantitative exposure assessment for the combustion of meat and bone meal derived from specified risk material in the context of BSE in Ireland

The probability and severity of an adverse event can be analyzed by quantitative exposure assessment (QEA). This methodology was applied to model the human health risks associated with the combustion of specified risk material (SRM) derived meat and bone meal (MBM) in a combustion facility. The identification of MBM and SRM as significant factors in the spread of bovine spongiform encephalopathy (BSE) has resulted in restrictions on their use and movement, and this has led to a requirement for alternative end-uses for these products. A stochastic (Latin Hypercube sampling) simulation model was developed to assess the exposure and hence the risks associated with the use of SRM-derived MBM in a combustion facility. The model simulates the potential infectivity pathways that SRM-derived MBM follows, including its production from animals potentially infected with sub-clinical BSE and subsequent processing of the material with segregation and heat treatments. A failure probability was included to take account of sub-optimal operating conditions. Two scenarios, reflecting the infectivity risk in different animal tissues as defined by the European Commission's scientific steering committee (SSC), were performed with 100,000 iterations of the model. Model results showed that the societal exposure to humans resulting from the combustion of SRM-derived MBM is extremely small (mean values ranging from 7.57 x 10(-6) ID50/year to 8.38 x 10(-5) ID50/year). The resulting societal risks are significantly less than the background societal risk of approximately 2.5 cases of sporadic CJD in Ireland each year. A sensitivity analysis revealed that the species barrier had a large impact on exposure calculations and hence should be the focus of further scientific investigation to reduce our uncertainty about this parameter. The model predicts that material spillage into untreated effluent represents the biggest risk to humans, indicating that efforts for risk mitigation should be focused on reducing the potential for spillage.     

作物叶片表面农药残留的便携式检测仪器的设计与试验

针对现有的农药残留检测仪器只能检测水溶液体系中的农药残留和检测对象较为单一的问题,该研究以不同植物叶片啶虫脒农药残留为研究对象,探究了利用荧光强度检测叶片表面农药残留的可行性,设计了一款叶片表面农药残留的便携式检测仪器。首先,通过啶虫脒农药叶片表面喷洒试验,采集叶片的荧光光谱并进行特征分析,发现啶虫脒农药的最佳激发波长和最佳发射波长分别为355和500 nm,从而确定光源和光电信号接收源的特征波长分别为350和500 nm。然后,通过获取最佳光源照射角度以及光照距离,优化光路结构减少叶片表面杂散光的干扰。同时,设计相关检测电路(光源电路、信号调理电路、控制电路等)测出表征反射光强度的电压值,构建电压值与农药残留值之间的线性方程,设计便携式检测仪对农药残留进行检测。结果表明:1)荧光强度与农药浓度在1～5 mg/L的范围内成正比;2)确定了检测仪器最佳光照角度为45°,光源和待测叶片之间的最佳垂直距离为3.46 cm;3)方程决定系数达到了0.875,均方根误差为0.405 mg/L。该研究所设计的便携式荧光光谱仪能够快速、准确、无损检测叶片表面农药残留。     

Concentrations of metals (zinc, copper, cadmium, and mercury) in three domestic ducks in France: Pekin, Muscovy, and Mule ducks

The role of different factors such as biological material (tissues, organs) and trophic condition (overfeeding or not) in the metal accumulation was studied in three genotypes of ducks (Pekin, Muscovy, and Mule) under breeding conditions. Results showed that overfeeding decreased the concentration in Cd, Cu, and Zn through the dilution process. In contrast, mercury concentration increased with this method. A relation between lipidic metabolism of genotypes and the distribution of this metal in biological material was found. Domestic ducks were little contaminated, but a low chronic contamination in Cd was observed, probably coming from the food. Due to the low levels of contamination observed in these breeding ducks, they can be considered as a good control for further contamination studies and comparison with accumulation levels recorded in the field. The impact of feeding condition on accumulation showed the importance of taking into account the life cycle of birds before studying their contamination and the impact of pollutants.     

Nondestructive application of laser-induced fluorescence spectroscopy for quantitative analyses of phenolic compounds in strawberry fruits (Fragaria x ananassa)

Laser-induced fluorescence spectroscopy (LIFS) was nondestructively applied on strawberries (EX = 337 nm, EM = 400-820 nm) to test the feasibility of quantitatively determining native phenolic compounds in strawberries. Eighteen phenolic compounds were identified in fruit skin by UV and MS spectroscopy and quantitatively determined by use of rp-HPLC for separation and diode-array or chemical reaction detection. Partial least-squares calibration models were built for single phenolic compounds by means of nondestructively recorded fluorescence spectra in the blue-green wavelength range using different data preprocessing methods. The direct orthogonal signal correction resulted in r (2) = 0.99 and rmsep < 8% for p-coumaroyl-glucose, and r (2) = 0.99 and rmsep < 24% for cinnamoyl-glucose. In comparison, the correction of the fluorescence spectral data with simultaneously recorded reflectance spectra did not further improve the calibration models. Results show the potential of LIFS for a rapid and nondestructive assessment of contents of p-coumaroyl-glucose and cinnamoyl-glucose in strawberry fruits.     

Assessing the risk of a 50-year-old dump site in the Baltic Sea by combining chemical analysis, bioaccumulation, and ecotoxicity

Purpose

During the late 1950s and early 1960s, industrial waste material highly enriched with various contaminants (e.g., heavy metals, polycyclic aromatic hydrocarbons (PAHs)) was dumped in the inner Bay of Mecklenburg, western Baltic Sea. Between 2002 and 2004, a research program was initiated using chemical analysis in combination with bioanalytical techniques to assess the extent and variability in contamination at this dump site (DS). The data were compared to a reference area (RS) with similar environmental conditions, which is representative of the western Baltic Sea.

Materials and methods

Twelve PAHs were investigated to assess their ecological hazard, as they were identified as major pollutants in the dumped material. In addition to analyzing the actual PAH contamination status in the sediments, PAHs measured in the soft tissue of Arctica islandica were also used as an indicator of contaminant bioaccumulation. A biotest battery was applied to determine the toxic effects of contaminants in the sediment.

Results and discussion

Significantly elevated PAH concentrations (sum of 12 PAHs) of ～3,000 ng g^?1 dw and higher bioaccumulation factors (BAFs) were determined in the soft body tissue of A. islandica collected at DS (t test, p?=?0.025). The results also showed that the sediment PAH contamination was significantly higher at DS (1,952–5,466 ng g^?1 dw) than at RS (1,384–2,315 ng g^?1 dw). The results revealed a major heterogeneity in the PAH concentration at DS due to an attempt to cover the toxic material with clean clay. This resulted in a more heterogeneous distribution of the dump material rather than covering it up completely. However, not all relevant contaminants were included in this study, not only because it is too costly to determine them all but also because unidentified contaminants present at concentrations below the limit of detection cannot be measured. Bioassays were used to fill this gap in the hazard assessment in a cost-effective way by investigating the possible effects of sediment contamination on benthic organisms. The results showed a high variability and magnitude of growth and luminescence inhibition. Bacterial contact tests with marine organisms showed a high toxicity response (>80 % inhibition) from DS sediments. In contrast, the luminescent bacteria test (Vibrio fischeri) showed equivalent effects of sediments from both DS and RS.

Conclusions

The spatial distribution of toxicity in DS, the bioaccumulation in mussels and the analytical evidence of PAH pollution clearly show that the dumped material still represents a potential risk even after 60 years.     

Assessment of Microbial Communities In Decomposition of Specified Risk Material Using a Passively Aerated Laboratory-Scale Composter

The occurrence of bovine spongiform encephalopathy (BSE) in Canada has resulted in the implementation of regulations to remove specified risk material (SRM) from the food chain. SRM includes the distal ileum of all cattle, and the skull, brain, trigeminal ganglia, eyes, palatine tonsils, and spinal cord and dorsal root ganglia of cattle ≥30 months of age. Composting may be a viable alternative to rendering for SRM disposal. In our study, two bulking agents, barley straw and wood shavings, were composted with beef manure along with SRM in passively aerated, laboratory-scale composters. Both composts heated rapidly, exceeding 55°C after 3 days with oxygen declining in the early composting stage with wood-shaving compost, but returning to near-original levels after 5 days. During composting the two matrices differed (P <0.05) only in water content, TC and bulk density. In the final compost, water content, TC and C/N ratio were higher (P < 0.05), while EC was lower (P < 0.05) in the wood shavings as compared to the straw compost. Approximately 50% of SRM was decomposed after 15 days of composting, with 30% of SRM being decomposed within the first 5 days. Phospholipid fatty acid (PLFA) profiles were used to characterize the microbial communities and showed that Gram positive bacteria were predominant in compost at day 5, a point that coincided with a rapid increase in temperature. Gram negative bacteria and anaerobes declined at day 5 but populations recovered by day 15. Fungi appeared to be suppressed as temperatures exceeded 55°C and did not appear to recover over the remainder of the composting period, with the exception of the straw compost at day 15. On day 5, Actinomycetes increased in the straw compost, but declined in the wood shavings compost, with this group increasing in both types of compost at day 15. Although temporal changes were evident, compost matrices or depth within the composter did not obviously influence microbial communities. Decomposition of SRM also did not differ between compost matrices or with depth in the composters. These results suggest that SRM decompose rapidly during composting and that both mesophilic and thermophilic microbial communities play a role in its decomposition.     

解释结构模型在中国疯牛病风险识别中的应用研究

疯牛病是一种严重危害牛类健康的疾病，它的感染因子可以传染给人类。因此这种疾病也威胁着人类的健康。我国目前还没有检测到疯牛病的存在。本文利用解释结构模型对疯牛病风险因素进行了定性评估。通过对模型进行运算求解得出清晰的结论：评估国反刍动物饲料中含有PrP^BS因子、评估国羊只患痒病和评估国牛患疯牛病包含在同一强连通域。